Biomedical subjects
M S Mitchell
Publications and source records attributed to M S Mitchell.
Characterization of uveal melanoma cell lines that grow as xenografts in rabbit eyes.
Two cell lines, OCM-1 and OCM-2, were established from biopsied specimens of choroidal melanomas of spindle B and mixed cell type morphologies. Both cell lines were phenotypically malignant. Karyotypic analyses revealed human chromosomes with a modal number of 95 and 85, respectively. These cell lines have been passaged for over 2 years and are essentially immortal. The cells grew without contact inhibition as monolayers in liquid culture or as clones in soft agar. Electron microscopy revealed melanoma cells containing premelanosomes and cultures free from contamination by fibroblasts. To categorize the morphologies of these cultured cells better by the Callender classification, they were grown as xenografts in the anterior chambers of rabbits immunosuppressed with daily i.m. doses of Cyclosporin A (10 mg/kg). Tumor plaques were detected after 10 days. The eyes were enucleated and fixed in formalin and stained with hematoxylin and eosin for histopathological evaluation. The xenograft from OCM-1 was found to consist predominantly of spindle B-type melanoma cells. In contrast, the xenograft from OCM-2 contained epithelioid, spindle B and clear cell ("balloon") melanoma cells. The ability of these cell lines to grow as xenografts confirmed their neoplastic origin. In fact, the types of the uveal melanoma cells in the xenografts resembled those in the original biopsied tumors. This suggests that the morphology of human uveal melanoma cells is an inherited trait and may be genetically fairly stable.
Combining chemotherapy with biological response modifiers in treatment of cancer.
Combinations of chemotherapeutic agents with various biological response modifiers ("biomodulators") in the treatment of cancer are now being tested. Since most (though not all) chemotherapy is immunosuppressive, the schedules by which the chemotherapeutic agents and the biomodulators are combined are of critical importance. This review summarizes the effects of chemotherapy on immunity and suggests the most logical ways in which these categories of drugs might be combined with biomodulators. Specific examples are also provided of combination therapies that have proved useful or that are of significant potential utility. An understanding of the types of action and the mechanisms of action of specific chemotherapeutic agents and specific biomodulators may help to achieve additive effects (possibly synergism) with these combinations. At the very least, that understanding will avoid the hazard of one modality's negating the effects of the other.
Active specific immunotherapy for melanoma: phase I trial of allogeneic lysates and a novel adjuvant.
A Phase I trial of active specific immunotherapy for melanoma was performed to measure the toxicity and immunological effects of the therapy. A mixture of mechanical lysates (homogenates) of two melanoma cell lines was injected together with a novel adjuvant, DETOX, into 22 patients. Several types of cell-mediated and humoral immunity to melanoma-associated antigens were measured serially. In the 17 patients with measurable disease, the sizes of lesions were also noted serially. At least six patients per group were injected s.c. with either 100, 200, or 400 antigenic units (approximately 10, 20, and 40 million tumor cell-equivalents) of the lysates mixed with 0.25 ml of DETOX s.c. on weeks 1, 2, 3, 4, and 6. Three patients at each dose level also received 300 mg/m2 of cyclophosphamide i.v. 4 days before the start of immunization. Evidence for successful immunization was obtained in 13 of the 22 patients. An increase in the frequency of peripheral blood cytolytic lymphocyte precursors reacting against melanoma cells occurred in 12 patients, as measured by a limiting dilution assay involving in vitro re-exposure to irradiated melanoma cells for 9-10 days. Eight of the 12 patients had received cyclophosphamide. By cold-target competition assays, these cytolytic lymphocytes appeared to be atypical T-cells, which recognized melanoma-associated antigens on several allogeneic lines without apparent major histocompatibility complex restriction. An increase in antibody titers against melanoma-associated antigens, measured by enzyme immunoassay, was found in five of 22 patients, and a change in delayed hypersensitivity against the melanoma lysate, in three patients. Responses were found at all three dosage levels of lysate, without an obvious dose optimum. No toxicity except minor local soreness was noted. Therefore, no maximum tolerable dose was defined. Five of 17 patients with measurable lesions had a remission of their melanoma, two complete and three partial, with three additional minor responses. A patient whose complete remission lasted 5.5 months, has no evidence of disease 22+ months after entry onto the study, with the aid of surgical resection of small s.c. recurrences on two separate occasions. Sites of regression included s.c. nodules, lymph nodes, and pulmonary nodules, with no responses in liver, adrenal gland, or bone. The patients who had an increase in cytolytic lymphocyte precursors comprised all eight with a clinical remission (five major, three minor). In contrast, none of the seven patients lacking an increase in cytotoxic lymphocytes had a clinical response.(ABSTRACT TRUNCATED AT 250 WORDS)
Biotechnology products in cancer therapy: a colloquium.
Explore the source record for details and available documents.
Studies on the relationship of human natural killer and lymphokine-activated killer cells with lysosomal staining and analysis of surface marker phenotypes.
We have previously demonstrated that natural killer (NK) cells are lysosome-rich and stain more intensely with lysosomotropic agents such as neutral red and quinacrine (Qu) than do non-NK cells. In this study we combined the quantitation of Qu staining with surface marker staining to define subpopulations of NK cells. While all NK activity was contained within the Qu+ population, most but not all NK cells expressed the surface marker CD16. A subpopulation of NK cells was found to be Qu+CD16- composed of medium- to large-sized cells with a granular appearance on Giemsa staining. Culture with interleukin-2 (IL-2) induced enhanced cytotoxicity in peripheral blood lymphocytes (PBL) against NK-sensitive and NK-resistant tumor cells. Like NK cells, these lymphokine-activated killer (LAK) cells were predominantly Qu+CD16+. However, some LAK cells were Qu+CD16-. The Qu+CD16+ cells were typical large granular lymphocytes (LGL). The Qu+CD16- cells were also large lymphocytes, more than 50% of which were proliferating. However, the granulation in some Qu+CD16- cells, as detected by Giemsa staining, was more prominent and numerous than others in the same population. No LAK activity was ever detected in Qu- cells, which were uniformly small lymphocytes. Quantitation of Qu staining in effector cells was therefore demonstrated to have a good correlation with NK and LAK functions, and with surface markers can help to characterize both types of cells. Moreover, these results indicate that both NK and LAK populations include a small subset of CD16- cells in each.
Effectiveness and tolerability of low-dose cyclophosphamide and low-dose intravenous interleukin-2 in disseminated melanoma [corrected].
We studied the effects on melanoma of low-dose recombinant interleukin-2 (IL-2) preceded by low-dose cyclophosphamide (CYC). Twenty-seven outpatients, aged 25 to 75 years, were treated with IL-2, 3.6 million U/m2 intravenously (IV), daily for five days on 2 successive weeks beginning three days after 350 mg/m2 of IV CYC. This schedule was repeated at least twice more at 1-week intervals. Six of 24 patients (25%) who received more than one 2-week cycle of treatment had a remission, one complete and five partial, with minor responses in eight others (33.3%). Three patients with rapidly progressive disease, who received only one cycle, were excluded from the analysis of response. The responses comprised remissions of liver metastases in two patients, one of them complete, two complete and two partial regressions of subcutaneous metastases, partial remission of lymph node metastases, and a partial remission of lung nodules. The mean duration of response exceeded 5 months, with two patients treated for greater than 1 year. Toxicity was moderate and controllable and only two patients required hospitalization, both overnight. Lymphokine-activated killer (LAK) cell activation was induced in 17 of the 24 patients, including all six responders, while none of seven patients without LAK activation had a remission. This regimen appeared to be as effective in melanoma as those involving ex vivo activation of LAK cells, and was generally tolerable to patients in all age groups.
Phase I study of recombinant beta-interferon given by four-hour infusion.
Twelve patients with advanced malignant disease were entered onto a Phase I study of escalating doses of beta-interferon serine given by 4-h i.v. infusion twice a wk. Three patients each were entered at starting doses of 0.01, 1, 10, and 30 million units (MU)/m2. Doses escalation within individual patients was allowed to a maximum dose of 400 MU/m2. Fever, chills, fatigue, and acral cyanosis were commonly seen and increased in frequency at higher doses. Myalgia, nausea, diarrhea, headache, and confusion were seen at lesser frequencies. Mild leukopenia, paresthesia, infusion site erythema, and hypotension were each seen in one patient. No conventional maximal tolerated dose could be defined, since several patients underwent escalation to the highest allowable dose and seemed to develop tolerance to acute toxicities. However, a maximal starting dose of 10 MU/m2 was identified, such that those begun at this level or below tolerated semiweekly dose escalation, while those begun at 30 MU/m2 could not tolerate continued therapy. Detectable serum interferon levels were noted during treatment at 10 and 30 MU/m2, the levels at which significant toxicity also first appeared. A maximal starting dose of 10 MU/m2, with gradual escalation as tolerance to side effects develops, is suggested if therapy with high-dose beta-interferon serine is given by 4-h infusion.
Altered antigenicity of human monoclonal antibodies derived from human-mouse heterohybridomas.
We have generated milligram quantities of human monoclonal antibodies (Hu-MAbs) in the ascites of pristane-primed nude mice injected with human-mouse heterohybridomas. After contaminating mouse immunoglobulins were removed by affinity chromatography, an enzyme immunosorbent assay (EIA) was used to measure the concentrations of human immunoglobulins. Ten different partially purified preparations were tested. The titration curves with all 5 IgG Hu-MAbs were unusual, reaching a plateau at a very low apparent maximum concentration of antibody. In contrast, the EIA yielded more usual titration curves and thus apparently more reliable estimates of the concentrations of 4 IgM and 1 IgA monoclonal antibodies. An analogous EIA for the quantitation of mouse IgG monoclonal antibodies also gave accurate estimates. To understand the nature of the discrepancy with human IgG, 5 Hu-MAbs of the 3 classes (2 IgG, 2 pentameric IgM and 1 IgA) were purified to homogeneity for a more detailed analysis. The inability to quantitate the human IgG monoclonal antibodies by EIA was not due to defective molecules, as shown by SDS polyacrylamide gel electrophoresis. The human IgG monoclonal antibodies were found to consist of intact heavy and light chains, as were the IgM and IgA antibodies. The possibility that the human IgG monoclonal antibodies differed antigenically from polyclonal IgG was explored by comparing the concentrations by EIA with the protein concentrations determined by absorbance at 280 nm. This analysis permitted a comparison of the detectability of antigenic determinants on Hu-MAbs with those on polyclonal Ig with goat antibodies to Ig or Ig subclass. The IgG monoclonal antibodies differed from polyclonal IgG in both their heavy and light chains. Goat antiserum monospecific for the gamma chain in fact underestimated the concentration by as much as one hundred-fold. IgM and IgA monoclonal antibodies were less antigenically distinct from their polyclonal counterparts even though their light chains were also underestimated, because goat monospecific antibodies were more efficient at recognizing their heavy chains. The molecular basis for the observed difference in antigenicity is not yet known. These findings have important implications for the analysis of the binding of IgG Hu-MAbs. A direct binding assay with the label directly conjugated to the Hu-MAb should be used in preference to an indirect assay with a labeled detecting antibody to maximize the sensitivity of the assay. The altered antigenicity of IgG Hu-MAbs may also imply decreased immunogenicity when they are given in vivo as carriers for radionuclides or cytotoxic antitumor materials.
Recombinant alfa interferon in renal cell carcinoma: a randomized trial of two routes of administration.
Ninety-seven patients with recurrent or metastatic renal cell carcinoma were randomized to receive recombinant interferon (IFN) alfa-2b (Intron A; Schering-Plough, Kenilworth, NJ) by either the subcutaneous (SC) or intravenous (IV) route. The SC dosage was 2 X 10(6) IU/m2 three times weekly, and the IV dose 30 X 10(6) IU/m2 for five consecutive days every 3 weeks. Dose escalation to a maximum of 10 X 10(6) IU/m2 SC and 50 X 10(6) IU/m2 IV was allowed for patients with minimal or absent toxicity. Five of 51 of the SC-treated patients (10%) and three of 46 of the IV-treated patients (7%) had a partial response (PR) or complete response (CR). Patients with prior nephrectomy, no prior treatment, and lack of bone metastases were most likely to respond, and a retrospective analysis of this subgroup revealed a 23% response rate. Achievement of response took from 3 weeks to 11 months, while response duration lasted from 3 to 31+ months. All responders had prior nephrectomy; six of eight had no prior chemotherapy or hormonal therapy; five had lung metastases, and none had bone metastases. Regardless of route, almost all patients developed a flu-like syndrome; however, grade 3 or greater toxicity was much more common for IV-treated patients. This trial demonstrates modest, but definite antitumor activity for recombinant interferon in advanced renal cell carcinoma. SC administration with lower dose and toxicity is as effective as treatment administered IV.
Human monoclonal antibodies that distinguish cutaneous malignant melanomas from benign nevi in fixed tissue sections.
Human monoclonal antibodies were generated by fusing a nonsecretory variant of murine myeloma cells with lymphocytes obtained from the lymph nodes of patients with metastatic cutaneous malignant melanoma. Two human IgG monoclonal antibodies, designated 2-139-1 and 6-26-3, were extensively studied for their patterns of binding to cells in 64 specimens of formalin-fixed, paraffin-embedded tissue sections. These comprised: 23 cutaneous and 2 ocular melanomas; 4 specimens of lentigo maligna; 27 benign nevi; 2 basal and 2 squamous cell neoplasms of the skin; and 4 specimens of normal skin. A direct avidin-biotin-immunoperoxidase staining method was used. Under these conditions, the antibodies reacted with variable intensity to all 18 primary cutaneous malignant melanomas, 5 metastatic cutaneous melanomas, and both ocular melanomas. Antibody 2-139-1 reacted with 1 of 4 specimens and 6-26-3 with 3 of 4 specimens of lentigo maligna. Two of 5 dysplastic nevi reacted with both antibodies, each with a smaller proportion of cells than with melanomas. There was no reactivity with the 22 other nevi representing a spectrum of histologic types or with normal melanocytes. Basal cell and squamous cell carcinomas of the skin also were not stained. These human monoclonal antibodies appear to be useful in distinguishing malignant melanomas from benign nevi, with the exception of dysplastic nevi, and from basal and squamous cancers of the skin in routinely prepared tissue sections. They may also help to identify the cytoplasmic antigens that are immunogenic in humans.
Monoclonal antibody T101 in T cell malignancies: a clinical, pharmacokinetic, and immunologic correlation.
Eight patients with cutaneous T cell lymphomas (CTCL) and five with various other T cell malignancies were treated with mouse monoclonal antibody (MoAb) T101. Doses of 1 to 500 mg were administered weekly over a two-hour period and resulted in one complete remission (convoluted T cell lymphoma) and one partial remission (CTCL). Remission duration was 6 weeks and 3 months, respectively. Frequent toxicities were pruritus, hives, flushing, and shortness of breath. Supraventricular arrhythmias and blood pressure instability were also observed. Complete targeting of peripheral blood T cells was achieved with 1 mg of MoAb in the nonleukemic patients (WBC less than 10,000/microL), and free, bioavailable antibody was present at the next (10-mg) dose level. Even higher doses resulted in substantial antibody excess that persisted for as long as 6 weeks. Serum concentrations of MoAb decreased with increasing number of peripheral blood T cells, and 25 to 35 mg of T101 were required for induction of antibody excess in leukemic patients. Excess antibody induced antigenic modulation, which was of consequence only if MoAb excess persisted to the next treatment. In the original treatment, the rapidly administered MoAb was able to target and remove peripheral blood T cells before the development of antigenic modulation. Antimouse antibodies developed in three patients. Their presence rendered further therapy ineffective and was associated with an anaphylactic reaction in one patient. Development of these antibodies could not be predicted by lymphoproliferative assays. In these assays, however, the T101 protein strongly stimulated the mononuclear cells of the patient who reached the only complete remission of this trial. Immunologic stimulation by the MoAb thus might have played a role in this patient's antitumor response. In summary, therapy with MoAb T101 was specific but only modestly efficacious. Rapid infusion of nonmodulating doses of antibody provided excellent targeting and removal of peripheral blood T cells and might be a valid approach in future trials with immunoconjugated T101.
Intratumoral Bacillus Calmette-Guérin immunotherapy prior to surgery for carcinoma of the lung: results of a prospective randomized trial.
A prospective randomized trial of preoperative intratumoral therapy with Bacillus Calmette-Guérin (BCG) was conducted in non-small cell lung cancer patients. Eighty-eight patients (48 BCG-treated and 40 control subjects) were entered into the study; three control subjects were removed from data analysis because histology revealed pathology other than non-small cell lung cancer. There were no differences between BCG-treated and control patients in sex, age, cigarettes smoked per day, pack-years of cigarette smoking, white blood cell count, or number of peripheral blood lymphocytes. Toxicity of BCG was limited to transient malaise and fever (average peak temperature, 38.7 degrees C). There was no significant difference in outcome (recurrence or survival) between BCG-treated and control groups with Stage I or Stage III tumors; there were too few Stage II tumors for separate statistical analysis. Outcome was not affected within or between the two treatment groups by tuberculin skin test status. Combining both treatment groups, Stage III patients had a worse outcome than did Stage I-II patients, non-squamous cell tumor patients (large cell and adenocarcinoma) had worse outcomes than did squamous cell tumor patients, and men had a worse outcome than women. We conclude that, although preoperative intratumoral BCG therapy is safe, it does not lengthen disease-free interval or prolong survival in patients with non-small cell lung cancer.
Human monoclonal antibodies directed against melanoma tumor-associated antigens.
To analyze the humoral immune response to melanoma, human-mouse hybridomas were generated by the fusions of regional lymph node lymphocytes of patients with the mouse myeloma cell line M5. Six stable hybridomas were cloned from six separate lymphocyte parents obtained from three patients. Ascites were obtained from nude mice after i.p. injection with cultured hybridoma cells. The monoclonal antibodies, four immunoglobulin Gs and two pentameric immunoglobulin Ms, were partially purified to remove mouse immunoglobulin and then conjugated to biotin for immunocytochemical and immunohistochemical studies. With the avidin:biotin:peroxidase complex method to detect and amplify binding by the biotin-conjugated human monoclonal antibodies, we found the six antibodies to be reactive against cytoplasmic determinants in five short-term melanoma cultures and formalin-fixed paraffin-embedded melanoma tumors from four patients. The antigenic target of the antibodies identified was not carcinoembryonic antigen. Two antibodies, 2-139-1 and 6-26-3, were studied in more detail. Each stained 25 of 25 specimens of melanomas. Little or no reactivity was detected against fixed sections of normal skin, which included tissues such as epidermis, dermis, monocytes, lymphocytes, and vascular endothelium. More striking was the absence of binding to melanocytes in the basal layer of the skin or to pigmented nevus cells. Both antibodies showed cross-reactivity against other tumors, in particular colonic and prostatic carcinomas. In the normal colon, reactivity was restricted to the surface of the columnar epithelium; no reactivity was detected against normal prostatic epithelium. Reactivity was also not observed against liver and lung. However, the epithelia of the renal tubules, pancreatic ducts, and salivary ducts were all reactive. These human monoclonal antibodies identify cytoplasmic melanoma-associated tumor antigens that appear different from the membrane antigens defined by serological approaches and by most mouse monoclonal antibodies.
Recombinant interferon alpha-2 (INTRON A) in a phase II study of renal cell carcinoma.
Because two of five patients with renal cell carcinoma in a Phase I study had partial response to recombinant alpha-2 interferon (IFN), we treated 26 patients with advanced renal cell carcinoma with a 3-month regimen of IFN. Patients were randomized to receive IFN either subcutaneously (2 X 10(6) IU/m2 3 times a week) or intravenously (3 X 10(7) IU/m2 for 5 consecutive days every 2-3 weeks). Patients whose disease was responding or stable were treated further, while those with progressive disease on subcutaneous treatment were offered intravenous therapy. Sites of metastasis included lung (14 patients), bone (7 patients), soft tissue (7 patients) and liver (2 patients). Twenty patients were evaluable for response. One patient had a partial response at the end of the third course of intravenous IFN and subsequently had complete disappearance of a 12 X 7 cm subcutaneous mass after the seventh course of treatment. The disease was stable in 13 patients including two minor responses, and six patients had progressive disease (5 with subcutaneous treatment; 1 with intravenous treatment) including one mixed response. All patients experienced early flu-like symptoms of fever, chills, and rigors during the first few days of treatment and most had mild to moderate fatigue. Three patients left the study because of fatigue, and one had an urticarial rash. From these results and our previous experience, it appears that IFN has activity against renal cell carcinoma with acceptable toxicity.
Inhibition of proliferation without affecting the generation of cytotoxicity in the human mixed lymphocyte reaction.
Phosphoramide mustard (PM) is considered to be the major tumoricidal metabolite of cyclophosphamide in vivo. The effects of this metabolite in vitro on several immune functions of human lymphocytes have been investigated. Very low concentrations (10(-7) to 10(-9) M) of PM added to lymphocyte cultures inhibited proliferation of the lymphocytes in response to mitogens and alloantigens. At these concentrations, inhibition of proliferation appeared to be due to a direct action of PM on the proliferative cells. Thus, concanavalin A-stimulated lymphocytes still acquired IL-2 receptors (Tac antigen) normally in the presence of PM (10(-6) to 10(-9) M). Only exceedingly high concentrations of PM (10(-5) M or greater) prevented the acquisition of Tac antigen. Similarly, the inhibition of proliferation was probably not related to endogenous IL-2 levels: addition of exogenous IL-2 to PM-containing cultures did not result in any restoration of proliferation. Further evidence that PM directly affected proliferative cells was that low concentrations of PM inhibited the proliferation of T cells continuously growing in IL-2. The exposure time to PM necessary for inhibition was essentially identical to those for lymphoproliferative responses to mitogens and alloantigens. Paradoxically, however, the generation of cytotoxic lymphocytes in mixed lymphocyte reactions (MLRs) and mixed lymphocyte tumor cell cultures (MLTCs) was very resistant to PM. In parallel MLRs and MLTCs the cytotoxic responses were resistant to approximately 1000-fold more PM than were the proliferative responses. Only at 10(-5) M PM were these inhibited. These data suggest that clonal expansion of cytotoxic lymphocytes or their precursors by proliferation is not an absolute requirement for the generation of cytolytic activity.
Effects of chemotherapeutic agents on the immune response. II.
Explore the source record for details and available documents.
Correlation between cytotoxic and suppressor activities of human pulmonary alveolar macrophages.
We have reported previously that pulmonary alveolar macrophages (PAMs) from individuals with lung cancer and active chronic pulmonary diseases were cytotoxic to tumor cells in vitro, whereas PAMs from normal individuals or patients with acute pulmonary disorders were noncytotoxic. In the present study, we evaluated 20 PAM preparations for both suppressor and cytotoxic functions to determine if PAMs could function as suppressor cells and, if so, whether a correlation between the two functions exists. Cytotoxicity was assessed in a 60-hr cytotoxicity assay against [3H]proline-prelabeled human melanoma target cells. More than 20% cytotoxicity was considered to be significant. Suppressor activity was measured by determining whether admixing PAMs at various ratios with autologous or allogeneic mononuclear cells could suppress concanavalin A-induced blastogenesis by T-lymphocytes. At least 50% suppression was considered to be significant. Of the 20 specimens evaluated, 13 were cytotoxic and 5 of these exhibited suppressor activity. None of the 7 noncytotoxic PAM preparations had suppressor activity. Suppression was nonspecific and not HLA restricted, since autologous and allogeneic mononuclear cells were inhibited to a similar extent. Suppression was probably not due to prostaglandin production by the PAMs since assays were performed under optimal conditions and required extremely high concentrations of prostaglandins. A significant correlation between suppressor and cytotoxic activity was found. Suppression was observed only with PAM specimens that were also highly cytotoxic to tumors, but not all cytotoxic PAM specimens were suppressive. Whether these actions reflect different levels of activation of PAMs or are the properties of different macrophage subsets remains to be clarified.